In mitosis, proper microtubule (MT) attachment to kinetochores is critical for faithful chromosome segregation. Our lab has shown that errors in kinetochore attachment occur at a high frequency in human tissue culture cells and these have to be corrected for accurate segregation. Budding yeast studies indicate a role for mitotic kinase Ipl1 (human Aurora B ortholog) in correcting MT misattachments at kinetochores. These studies show that Ipl1 phosphorylates the highly conserved outer kinetochore component Ndc80 (human Hec1 ortholog) and a MT-associated protein Dam1 (no known human ortholog). Genetic and mutagenesis studies indicate that Aurora B phosphorylation of these substrates weakens kinetochore interactions with kinetochore MTs (kMTs). Preliminary data from my yeast studies suggest Aurora B regulates another MT-associated protein Dim1 (human EB1). EB1 proteins are known to bind and promote stability of plus-end kMTs. I propose to investigate how human Hec1 and EB1 function are regulated by aurora B at the vertebrate kMT interface. I will examine the consequences of presence or absence of Aurora B phosphorylation of these k-MT substrates in mammalian cells.